Arsenic poisoning, or arsenicosis, is a worldwide threat to public health, leading to a variety of human diseases, including cancer. The microbial community (microbiome) of the human GI tract (GIT) has been implicated as a significant influence on host exposure to toxic xenobiotics, including arsenic-containing compounds (arsenicals), but the individual roles of host vs. microbiome in arsenic biotransformation have not been clearly defined. Our preliminary data in mice strongly suggest that the microbiome decreases arsenic toxicity in the host. The broad, long-term objective of this research is to bette understand the functional components of the human microbiome that impact As-transformations in the GIT that can then be manipulated as prophylactic and/or detoxifying agents for use as novel treatment and prevention strategies against human arsenicosis. This research addresses the microbiome's role in human exposure to an environmental toxin and so specifically addresses a strategic theme (Exposure Research) and a specific strategic goal (Goal 4, part b) of the National Institute of Environmental Health Sciences (NIEHS). As an initial step toward defining the role of the human microbiome in arsenicosis, Specific Aim 1 will establish the baseline production of arsenicals in germ free mice and germ free mice colonized with a human microbiome (humanized mice). Germ free mice are completely sterile and so arsenical production in these arsenic-exposed animals will be due to host metabolism alone. In contrast, arsenical production in humanized mice will reflect the net influence of host and microbe, thereby allowing a comparison of their individual roles. As the next step forward in defining the role of the human microbiome in arsenicosis, Specific Aim 2 will directly quantify the influence of microbially- produced, arsenic-active enzymes in the gastrointestinal tract on arsenical levels in gnotobiotic mice. In this part of the project, germ free mice will be mono-associated with genetically defined strains of Escherichia coli that have been shown previously to metabolize arsenic in specific ways. Arsenical production will be quantified from temporally collected mouse tissues and excretia by state-of-the-art methodology using high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICPMS) and corresponding temporal microbiome dynamics will be tracked using 16S rRNA encoding gene metagenomic sequencing. These data will be analyzed together to provide statistical support to and experimental evidence for the in vivo transformation of arsenic by the human GIT microbiome.